PROJECT SUMMARY/ABSTRACT: This program aims to develop a novel storage method for human ovarian tissue and whole ovaries, which can also be applied to testicular tissue and gonadal preservation as well as a variety of cells, tissues, and whole organs. This multi-pronged approach builds on recent advances in machine perfusion, nature-inspired cytoprotection strategies, and non-toxic cryoprotectant solutions, combining them with our novel method for constant-volume, pressure assisted cooling. Our method promises to achieve vitrification of living tissues without the aid of toxic concentrations of cryoprotectants, in a system at thermodynamic equilibrium and potentially at temperatures as high as -70C. It ameliorates or entirely circumvents many of the limitations of conventional cryopreservation methods for large tissues, including damaging ice crystallization, eleterious changes in solute concentrations, and volumetric changes. This approach has the potential to enable indefinite banking of ovarian tissues, whole ovaries, and other living materials while dramatically limiting tissue injury currently associated with ex-vivo storage. The technical objective of this Phase 1 proposal is to develop an optimized cocktail and protocol for preservation and indefinite banking of ovarian tissue strips. We will develop our method across three specific aims. In Aim 1, will use well established methods developed in one of our labs for comprehensive thermodynamic characterization of novel, nature-inspired cryoprotectant solutions in an isochoric (constant- volume) system; this will allow us to select solutions that allow for pressure-assisted vitrification of living materials at low to intermediate pressures and much higher temperatures than traditionally needed for ice-free cryopreservation. In Aim 2, we select the most effective cryoprotectant solutions established in Aim 1 to optimize our ?high sub-zero vitrification? in a cell-based model for ovarian tissue consisting of human primary ovarian epithelial cells, human primary ovarian granulosa cells, and human endothelial cells; using high- throughput combinatorial testing, we will select protocols which maximize cell viability and improve on current preservation standards. In Aim 3, we will test our optimized protocols for the preservation of human ovarian cortical tissue strips, along with one of the most successful protocols for ovarian tissue freezing and for ovarian tissue vitrification, respectively; we will additionally test the augmentation of these methods with subnormothermic machine perfusion, enabling further reduction of ischemic tissue injury. Success of these novel approaches individually or in combination will likely enable breakthroughs in clinical oncofertility and biopreservation.